Biotinylated Probes for the Analysis of Protein Modification by Electrophiles

  • Simona G. Codreanu
  • Hye-Young H. Kim
  • Ned A. Porter
  • Daniel C. Liebler
Part of the Methods in Molecular Biology book series (MIMB, volume 803)


Formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu+-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.

Key words

Electrophile Click chemistry Protein adducts Lipid oxidation Photocleavable Biotin Shotgun proteomics 


  1. 1.
    West, J. D., and Marnett, L. J. (2006) Endogenous Reactive Intermediates as Modulators of Cell Signaling and Cell Death. Chem. Res. Toxicol. 19, 173–194.PubMedCrossRefGoogle Scholar
  2. 2.
    Mashima, R., Witting, P. K., and Stocker, R. (2001) Oxidants and antioxidants in atherosclerosis. Curr. Opin. Lipidol. 12, 411–418.PubMedCrossRefGoogle Scholar
  3. 3.
    Ames, B. N. (2001) DNA damage from micronutrient deficiencies is likely to be a major cause of cancer. Mutation Research 475, 7–20.PubMedCrossRefGoogle Scholar
  4. 4.
    Beckman, K. B., and Ames, B. N. (1998) The Free Radical Theory of Aging Matures. Physiol. Rev. 78, 547–581.PubMedGoogle Scholar
  5. 5.
    Codreanu, S. G., Zhang, B., Sobecki, S. M., Billheimer, D. D., and Liebler, D. C. (2009) Global Analysis of Protein Damage by the Lipid Electrophile 4-Hydroxy-2-nonenal. Mol. Cell. Proteomics 8, 670–680.PubMedCrossRefGoogle Scholar
  6. 6.
    Vila, A., Tallman, K. A., Jacobs, A. T., Liebler, D. C., Porter, N. A., and Marnett, L. J. (2008) Identification of Protein Targets of 4-Hydroxynonenal Using Click Chemistry for ex Vivo Biotinylation of Azido and Alkynyl Derivatives. Chem. Res. Toxicol. 21, 432–444.PubMedCrossRefGoogle Scholar
  7. 7.
    Kim, H.-Y. H., Tallman, K. A., Liebler, D. C., and Porter, N. A. (2009) An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease. Mol. Cell. Proteomics 8, 2080–2089.PubMedCrossRefGoogle Scholar
  8. 8.
    Tallman, K. A., Kim, H.-Y. H., Ji, J.-X., Szapacs, M. E., Yin, H., McIntosh, T. J., Liebler, D. C., and Porter, N. A. (2007) Phospholipid-Protein Adducts of Lipid Peroxidation: Synthesis and Study of New Biotinylated Phosphatidylcholines. Chem. Res. Toxicol. 20, 227–234.PubMedCrossRefGoogle Scholar
  9. 9.
    Tabb, D. L., Fernando, C. G., and Chambers, M. C. (2007) MyriMatch: Highly Accurate Tandem Mass Spectral Peptide Identification by Multivariate Hypergeometric Analysis. J. Proteome Res. 6, 654–661.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang, B., Chambers, M. C., and Tabb, D. L. (2007) Proteomic Parsimony through Bipartite Graph Analysis Improves Accuracy and Transparency. J. Proteome Res. 6, 3549–3557.PubMedCrossRefGoogle Scholar
  11. 11.
    Elias, J. E., and Gygi, S. P. (2007) Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Meth. 4, 207–214.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Simona G. Codreanu
    • 1
  • Hye-Young H. Kim
    • 1
  • Ned A. Porter
    • 1
  • Daniel C. Liebler
    • 1
  1. 1.Vanderbilt University School of MedicineNashvilleUSA

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